Botulinum toxin compositions and methods

ABSTRACT

Disclosed herein are methods of using extracellular matrix digesting enzymes and neurotoxins, such as a Clostridial neurotoxins, to treat various medical conditions, such as overactive bladder, benign prostatic hyperplasia, hyperhidrosis, for example.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/890,052, filed Feb. 15, 2007, the contents of which are herebyincorporated by reference in their entirety.

BACKGROUND

The present invention relates to the use of extracellular matrixdigesting enzymes and neurotoxins to treat various medicalconditions/disorders, such as overactive bladder, urinary incontinencedue to overactive bladder or unstable detrusor sphincter, benignprostatic hyperplasia and associated bladder voiding complications,urinary retention that is secondary to having a spastic sphincter or ahypertrophied bladder neck, neurogenic bladder dysfunction (e.g.secondary to for example, Parkinson's disease, spinal cord injury,stroke or multiple sclerosis) and hyperhidrosis.

Neurotoxins, and in particular botulinum toxins, are increasinglyfinding useful application in treating various medical conditions. Suchtreatments are typically focally delivered via injections that penetratethe skin or organ lining. This can lead to difficulty in delivering thetreatment due to complications from needle penetration, patient concernssuch as needle phobia, pain and physician training issues. Neurotoxinssuch as botulinum toxin are gaining significant application in thetreatment of several urological conditions including overactive bladder(OAB) and detrusor hyperreflexia (DH) which cause bothersome symptomssuch as voiding urgency, excessive voiding frequency and incontinence,for example. A detailed discussion of the use and techniques forutilizing botulinum toxin to treat overactive bladder can be found in“Botulinum toxin for the treatment of idiopathic and neurogenicoveractive bladder: State of the art” Nitti Victor W. Rev Urol 2006;8(4):198-208. As detailed therein, botulinum toxin is injected into thebladder wall and the number of injections (between 15 to 50 injectionsof 100 to 1000 units of botulinum toxin type A and 10 injections of 5000units botulinum toxin type B) depends on the well known effect andpotency difference between the serotype of botulinum toxin utilized, aswell as the amount of total toxin and dilution of toxin utilized, asdetailed in therein and known in the art.

Incontinence, one symptom of various urologic disorders, includes urgeincontinence and stress incontinence. Urge incontinence involves astrong, sudden need to urinate, followed by inappropriate bladdercontraction, which then results in leakage. What is troublesome is thatit is often the case that these contractions occur regardless of theamount of urine that is in a sufferer's bladder, that is, the bladderdoes not necessarily have to be so full and under pressure from urinecontained therein to result undesirable leakage. Urge incontinence canbe a result of neurological injuries (such as spinal cord injury orstroke), neurological diseases (such as multiple sclerosis), infection,bladder cancer, bladder stones, bladder inflammation, or bladder outletobstruction, for example. While these conditions can be found both inmen and women, men have an additional burden in that urge incontinencemay also be due to neurologic disease or bladder changes caused bybenign prostatic hypertrophy (BPH) or bladder outlet obstruction from anenlarged prostate, for example.

Stress incontinence is an involuntary loss of urine that occurs duringphysical activity, such as coughing, sneezing, laughing, or exercise. Aperson can suffer from one or both types of incontinence, and whensuffering from both, it is called mixed incontinence. Despite all of theknowledge associated with incontinence, the majority of cases of urgeincontinence are idiopathic, which means a specific cause cannot beidentified. Urge incontinence may occur in anyone at any age, and it ismore common in women and the elderly.

The detrusor of the bladder is the muscle that expels urine from thebladder. Consequences of detrusor hyperreflexia include poor bladdercompliance, high intravesical pressure, and reduction in bladdercapacity, all of which may result in deterioration of the upper urinarytract.

It is thought that botulinum toxin exerts its effect on bladderhyperactivity by paralyzing the detrusor muscle in the bladder wall orpossibly impacting afferent pathways in the bladder and reducing sensoryreceptors in suburothelial nerves. These effects possibly account forthe improvement in urinary incontinence, bladder capacity and reductionin bladder detrusor pressures that are seen when the bladder walls areinjected with botulinum toxins. Examples of botulinum toxin use to treatvarious urologic disorders can be found in “Botulinum Toxin Treatment ofSpastic Bladder”, by Dott, C. et al., U.S. Patent App. Publication No.US 2007/0275110A1 and “Methods for the use of neurotoxin in thetreatment of urologic disorders”, by Doshi, R., U.S. Patent App.Publication No. 2004/0067235A1, both herein incorporated by reference.Other known potential urological applications for neurotoxins includethe treatment of a variety of disorders of the prostate including benignprostatic hyperplasia (BPH), prostatitis, and prostate cancer (see,e.g., U.S. Pat. No. 6,365,164, herein incorporated by reference in itsentirety.)

To date, botulinum toxin has shown promising early results for treatmentof lower urinary tract symptoms including obstructive and irritativevoiding symptoms attributed to BPH. Both subjective (symptoms) andobjective (flow rates) improvements have been observed. The prostate isa partially glandular and partially fibromuscular gland of the malereproductive system. During aging, the prostate tends to enlarge(hypertrophy). This prostatic enlargement can lead to urethralobstruction and voiding dysfunction. This is because the urethra passesthrough the prostate (prostatic urethra) as it leads to the externalurethral orifice. A detailed discussion of prostate anatomy (includinglobes, stroma, nerve fiber types and innervation) can be found inpublished U.S. patent application Ser. No. 09/978,982, filed Oct. 15,2001, and entitled “Use of neurotoxin therapy for treatment of urologicand related disorders”, U.S. Published Patent Application No.20020025327 A1, herein incorporated by reference in its entirety, inaddition to standard anatomy texts.

Botulinum toxin is thought to affect nerve terminals in the prostate andthe release of neurotransmitters including acetylcholine, sensoryneuropeptides, and noradrenalin. These effects may alter neural controlwithin the prostate. Preliminary reports suggest that botulinum toxinmay also have a role in the management of prostate cancer, possibly byinhibiting inflammation and the down regulation of COX-2 expression.

The large size of the botulinum toxin molecule can limit its ability todiffuse, and thus prohibits it from reaching both afferent and efferentnerve fibers. As a result, current methods of administration for OAB,for example, require many injections (typically 20 to 50) of botulinumtoxin into the bladder muscle wall or into the prostate. Other examplesof botulinum toxin uses includes the treatment of chronic migraine withbotulinum toxin, which requires approximately 30 injections into thehead and neck musculature, and axillary hyperhidrosis, which requiresnumerous injections to the dermal skin layer in the axilla (typicallyanywhere from 10 to 40 injections per axilla, depending on the severityof the condition, area overproducing sweat, size of the patient andconcentration, amount and type of botulinum toxin used).

The genus Clostridium has more than one hundred and twenty sevenspecies, grouped according to their morphology and functions. Theanaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

About 50 picograms of a commercially available botulinum toxin type A (apurified neurotoxin complex available from Allergan, Inc., of Irvine,Calif. under the tradename BOTOX® in 100 unit vials) is a LD₅₀ in mice(i.e. 1 unit). One unit of BOTOX® contains about 50 picograms (about 56attomoles) of botulinum toxin type A complex. Interestingly, on a molarbasis, botulinum toxin type A is about 1.8 billion times more lethalthan diphtheria, about 600 million times more lethal than sodiumcyanide, about 30 million times more lethal than cobra toxin and about12 million times more lethal than cholera. Singh, Critical Aspects ofBacteria/Protein Toxins, pages 63-84 (chapter 4) of Natural Toxins II,edited by B. R. Singh et al., Plenum Press, New York (1976) (where thestated LD₅₀ of botulinum toxin type A of 0.3 ng equals 1 unit iscorrected for the fact that about 0.05 ng of BOTOX® equals 1 unit). Oneunit (U) of botulinum toxin is defined as the LD₅₀ upon intraperitonealinjection into female Swiss Webster mice weighing 18 to 20 grams each.

Seven immunologically distinct botulinum neurotoxins have beencharacterized, these being respectively botulinum neurotoxin serotypesA, B, C₁, D, E, F and G, each of which is distinguished byneutralization with type-specific antibodies. The different serotypes ofbotulinum toxin vary in the animal species that they affect and in theseverity and duration of the paralysis they evoke. For example, it hasbeen determined that botulinum toxin type A is 500 times more potent, asmeasured by the rate of paralysis produced in the rat, than is botulinumtoxin type B. Additionally, botulinum toxin type B has been determinedto be non-toxic in primates at a dose of 480 U/kg which is about 12times the primate LD₅₀ for botulinum toxin type A. Moyer E et al.,Botulinum Toxin Type 8: Experimental and Clinical Experience, beingchapter 6, pages 71-85 of “Therapy With Botulinum Toxin,” edited byJankovic, J. et al. (1994), Marcel Dekker, Inc. Botulinum toxinapparently binds with high affinity to cholinergic motor neurons, istranslocated into the neuron, and blocks the release of acetylcholine.Additional uptake can take place through low affinity receptors, as wellas by phagocytosis and pinocytosis.

Regardless of stereotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least three steps or stages. Inthe first step of the process, the toxin binds to the presynapticmembrane of the target neuron through a specific interaction between theheavy chain, H chain, and a cell surface receptor; the receptor isthought to be different for each type of botulinum toxin and for tetanustoxin. The carboxyl end segment of the H chain, H_(C), appears to beimportant for targeting of the toxin to the cell surface. In the secondstep, the toxin crosses the plasma membrane of the poisoned cell. Thetoxin is first engulfed by the cell through receptor-mediatedendocytosis, and an endosome containing the toxin is formed. The toxinthen escapes the endosome into the cytoplasm of the cell. This step isthought to be mediated by the amino end segment of the H chain, H_(N),which triggers a conformational change of the toxin in response to a pHof about 5.5 or lower. Endosomes are known to possess a proton pumpwhich decreases intra-endosomal pH. The conformational shift exposeshydrophobic residues in the toxin, which permits the toxin to embeditself in the endosomal membrane. The toxin (or at a minimum the lightchain) then translocates through the endosomal membrane into thecytoplasm.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the heavy chain, Hchain, and the light chain, L chain. The entire toxic activity ofbotulinum and tetanus toxins is contained in the L chain of theholotoxin; the L chain is a zinc (Zn²⁺) endopeptidase which selectivelycleaves proteins essential for recognition and docking ofneurotransmitter-containing vesicles with the cytoplasmic surface of theplasma membrane, and fusion of the vesicles with the plasma membrane.Tetanus neurotoxin, botulinum toxin types B, D, F, and G, causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the VAMPpresent at the cytoplasmic surface of the synaptic vesicle is removed asa result of any one of these cleavage events. Botulinum toxin serotype Aand E cleave SNAP-25. Botulinum toxin serotype C₁ was originally thoughtto cleave syntaxin, but was found to cleave syntaxin and SNAP-25. Eachof the botulinum toxins specifically cleaves a different bond, exceptbotulinum toxin type B (and tetanus toxin) which cleave the same bond.Each of these cleavages block the process of vesicle-membrane docking,thereby preventing exocytosis of vesicle content.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletal muscles(i.e. motor disorders). Almost twenty years ago, in 1989, a botulinumtoxin type A complex was approved by the U.S. Food and DrugAdministration for the treatment of blepharospasm, strabismus andhemifacial spasm. Subsequently, a botulinum toxin type A was alsoapproved by the FDA for the treatment of cervical dystonia and for thetreatment of glabellar lines, and a botulinum toxin type B was approvedfor the treatment of cervical dystonia. Non-type A botulinum toxinserotypes apparently have a lower potency and/or a shorter duration ofactivity as compared to botulinum toxin type A. Clinical effects ofperipheral intramuscular botulinum toxin type A are usually seen withinone week of injection. The typical duration of symptomatic relief from asingle intramuscular injection of botulinum toxin type A averages aboutthree months, although significantly longer periods of therapeuticactivity have been reported.

Although all the botulinum toxin serotypes apparently inhibit release ofthe neurotransmitter acetylcholine at the neuromuscular junction, theydo so by affecting different neurosecretory proteins and/or cleavingthese proteins at different sites. For example, botulinum types A and Eboth cleave the 25 kiloDalton (kD) synaptosomal associated protein(SNAP-25), but they target different amino acid sequences within thisprotein. Botulinum toxin types B, D, F and G act on vesicle-associatedprotein (VAMP, also called synaptobrevin), with each serotype cleavingthe protein at a different site. Finally, botulinum toxin type C₁ hasbeen shown to cleave both syntaxin and SNAP-25. These differences inmechanism of action may affect the relative potency and/or duration ofaction of the various botulinum toxin serotypes. Apparently, a substratefor a botulinum toxin can be found in a variety of different cell types.See e.g. Biochem J 1; 339 (pt 1):159-65.1999, and MovDisord,10(3):376:1995 (pancreatic islet B cells contains at least SNAP-25 andsynaptobrevin).

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ are apparentlyproduced as only a 700 kD or 500 kD complex. Botulinum toxin type D isproduced as both 300 kD and 500 kD complexes. Finally, botulinum toxintypes E and F are produced as only approximately 300 kD complexes. Thecomplexes (i.e. molecular weight greater than about 150 kD) are believedto contain a non-toxin hemaglutinin protein and a non-toxin andnon-toxic nonhemaglutinin protein. These two non-toxin proteins (whichalong with the botulinum toxin molecule comprise the relevant neurotoxincomplex) may act to provide stability against denaturation to thebotulinum toxin molecule, and protection against digestive acids whentoxin is ingested. Additionally, it is possible that the larger (greaterthan about 150 kD molecular weight) botulinum toxin complexes may resultin a slower rate of diffusion of the botulinum toxin away from a site ofintramuscular injection of a botulinum toxin complex.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine (Habermann E., et al., Tetanus Toxin and Botulinum A andC Neurotoxins Inhibit Noradrenaline Release From Cultured Mouse Brain JNeurochem 51(2); 522-527:1988)), CGRP, substance P, and glutamate(Sanchez-Prieto, J., et al., Botulinum Toxin A Blocks GlutamateExocytosis From Guinea Pig Cerebral Cortical Synaptosomes, Eur J.Biochem 165; 675-681:1897). Thus, when adequate concentrations are used,stimulus-evoked release of most neurotransmitters is blocked bybotulinum toxin. See e.g. Pearce, L. B., Pharmacologic Characterizationof Botulinum Toxin For Basic Science and Medicine, Toxicon 35(9); 1373-1 412 at 1393; Bigalke H., et al., Botulinum A Neurotoxin InhibitsNon-Cholinergic Synaptic Transmission in Mouse Spinal Cord Neurons inCulture, Brain Research 360; 318-324:1985; Habermann E., Inhibition byTetanus and Botulinum A Toxin of the release of [3H] Noradrenaline and[3H]GABA From Rat Brain Homogenate, Experientia 44; 224-226: 1988,Bigalke H., et al., Tetanus Toxin and Botulinum A Toxin Inhibit Releaseand Uptake of Various Transmitters, as Studied with ParticulatePreparations From Rat Brain and Spinal Cord, Naunyn-Schmiedeberg's ArchPharmacol 31 6; 244-251:1 981, and; Jankovic J. et al., Therapy WithBotulinum Toxin, Marcel Dekker, Inc., (1994), page 5.

Botulinum toxin type A can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and G cantherefore be recovered from bacterial cultures in predominantly theiractive form. In contrast, botulinum toxin serotypes C₁, D and E aresynthesized by nonproteolytic strains and are therefore typicallyunactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains and therefore can berecovered in either the active or inactive form. However, even theproteolytic strains that produce, for example, the botulinum toxin typeB serotype, only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin, is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B, as compared tobotulinum toxin type A (and thus the routine use of many thousands ofunits of botulinum toxin type B, as known in the art, see e.g.“Long-term safety, efficacy, and dosing of botulinum toxin type B(MYOBLOC®) in cervical dystonia (CD) and other movement disorders” KumarR and Seeberger LC. Mov Disord 2002; 17(Suppl 5):S292-S293). Thepresence of inactive botulinum toxin molecules in a clinical preparationwill contribute to the overall protein load of the preparation, whichhas been linked to increased antigenicity, without contributing to itsclinical efficacy. Additionally, it is known that botulinum toxin type Bhas, upon intramuscular injection, a shorter duration of activity and isalso less potent than botulinum toxin type A at the same dose level.

High quality crystalline botulinum toxin type A can be produced from theHall A strain of Clostridium botulinum with characteristics of >3×10⁷U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern of bandingon gel electrophoresis. The known Schantz process can be used to obtaincrystalline botulinum toxin type A, as set forth in Schantz, E. J., etal, Properties and use of Botuilnum toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56; 80-99:1992. Generally, thebotulinum toxin type A complex can be isolated and purified from ananaerobic fermentation by cultivating Clostridium botulinum type A in asuitable medium. The known process can also be used, upon separation outof the non-toxin proteins, to obtain pure botulinum toxins, such as forexample: purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; and purified botulinum toxin type F with an approximately 155kD molecular weight with a specific potency of 1-2×10⁷ LD₅₀ U/mg orgreater.

Botulinum toxins and/or botulinum toxin complexes can be obtained fromList Biological Laboratories, Inc., Campbell, Calif.; the Centre forApplied Microbiology and Research, Porton Down, U. K.; Wako (Osaka,Japan), Metabiologics (Madison, Wis.) as well as from Sigma Chemicals ofSt Louis, Mo. Pure botulinum toxin can also be used to prepare apharmaceutical composition for use in accordance with the presentdisclosure.

As with enzymes generally, the biological activities of botulinum toxins(which are intracellular peptidases) is dependant, at least in part,upon their 3-dimensional conformation. Thus, botulinum toxin type A isdetoxified by heat, various chemicals, surface stretching, and surfacedrying. Additionally, it is known that dilution of the toxin complexobtained by the known culturing, fermentation and purification to themuch lower toxin concentrations used for pharmaceutical compositionformulation results in rapid detoxification of the toxin unless asuitable stabilizing agent is present. Dilution of the toxin frommilligram quantities to a solution containing nanograms per milliliterpresents significant difficulties because of the rapid loss of specifictoxicity upon such great dilution. Since the toxin may be used months oryears after the toxin containing pharmaceutical composition isformulated, the toxin can be stabilized with a stabilizing agent such asalbumin and gelatin.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, albumin and sodium chloride packaged in sterile,vacuum-dried form. Botulinum toxin type A is made from a culture of theHall strain of Clostridium botulinum grown in a medium containing N-Zamine and yeast extract. The botulinum toxin type A complex is purifiedfrom the culture solution by a series of acid precipitations to acrystalline complex consisting of the active high molecular weight toxinprotein and an associated hemagglutinin protein. The crystalline complexis re-dissolved in a solution containing saline and albumin and sterilefiltered (0.2 microns) prior to vacuum-drying. The vacuum-dried productis stored in a freezer at or below −5° C. BOTOX® can be reconstitutedwith sterile, non-preserved saline prior to intramuscular injection.Each vial of BOTOX® contains about 100 U of Clostridium botulinum toxintype A purified neurotoxin complex, 0.5 milligrams of human serumalbumin and 0.9 milligrams of sodium chloride in a sterile, vacuum-driedform without a preservative.

To reconstitute vacuum-dried BOTOX®, sterile normal saline without apreservative (0.9% Sodium Chloride Injection) is used by drawing up theproper amount of diluent in the appropriate size syringe. Since BOTOX®may be denatured by bubbling or similar violent agitation, the diluentis gently injected into the vial. For sterility reasons BOTOX® ispreferably administered within four hours after the vial is removed fromthe freezer and reconstituted. During these four hours, reconstitutedBOTOX® can be stored in a refrigerator at about 2° C. to about 8° C.Reconstituted, refrigerated BOTOX® has been reported to retain itspotency for at least about two weeks (Neurology, 48:249-53, 1997). Ithas been reported that botulinum toxin type A has been used in clinicalsettings as follows:

(1) about 75-125 U of BOTOX® per intramuscular injection (multiplemuscles) to treat cervical dystonia;(2) 5-10 U of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercilii muscle);(3) about 30-80 U of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle;(4) about 1-5 U per muscle of intramuscularly injected BOTOX® to treatblepharospasm by injecting the lateral pre-tarsal orbicularis oculimuscle of the upper lid and the lateral pre-tarsal orbicularis oculi ofthe lower lid;(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 U of BOTOX®, the amount injectedvarying based upon both the size of the muscle to be injected and theextent of muscle paralysis desired (i.e. amount of diopter correctiondesired);(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:(a) flexor digitorum profundus: 7.5 U to 30 U(b) flexor digitorum sublimus: 7.5 U to 30 U(c) flexor carpi ulnaris: 10 U to 40 U(d) flexor carpi radialis: 15 U to 60 U(e) biceps brachii: 50 U to 200 U. Each of the five indicated muscleshas been injected at the same treatment session, so that the patientreceives from 90 U to 360 U of upper limb flexor muscle BOTOX® byintramuscular injection at each treatment session;(7) to treat migraine, pericranial (injected symmetrically intoglabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX®has showed significant benefit as a prophylactic treatment of migrainecompared to vehicle as measured by decreased measures of migrainefrequency, maximal severity, associated vomiting and acute medicationuse over the three month period following the 25 U injection.

It is known that botulinum toxin type A can have an efficacy for up to12 months (European J. Neurology 6 (Supp 4): S111-S1150: 1999), and insome circumstances for as long as 27 months, when used to treat glands,such as in the treatment of hype rhydrosis. See e.g. Bushara K.,Botulinum toxin and rhinorrhea, Otolaryngol Head Neck Surg 1996;114(3):507, and The Laryngoscope 109:1344-1346:1999. However, the usualduration of effect of an intramuscular injection of BOTOX® is typicallyabout 3 to 4 months.

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes. Twocommercially available botulinum type A preparations for use in humansare BOTOX® available from Allergan, Inc., of Irvine, Calif., andDYSPORT® available from Beaufour Ipsen, Porton Down, England. Abotulinum toxin type B preparation (MYOBLOC®) is available from ElanPharmaceuticals of San Francisco, Calif.

A botulinum toxin has also been proposed for or has been used to treatotitis media of the ear (U.S. Pat. No. 5,766,605), inner ear disorders(U.S. Pat. Nos. 6,265,379; 6,358,926), tension headache, (U.S. Pat. No.6,458,365), migraine headache pain (U.S. Pat. No. 5,714,468),post-operative pain and visceral pain (U.S. Pat. No. 6,464,986), hairgrowth and hair retention (U.S. Pat. No. 6,299,893), psoriasis anddermatitis (U.S. Pat. No. 5,670,484), injured muscles (U.S. Pat. No.6,423,319) various cancers (U.S. Pat. No. 6,139,845), smooth muscledisorders (U.S. Pat. No. 5,437,291), and neurogenic inflammation (U.S.Pat. No. 6,063,768). Controlled release toxin implants are known (seee.g. U.S. Pat. Nos. 6,306,423 and 6,312,708) as is transdermal botulinumtoxin administration (U.S. patent application Ser. No. 10/194,805).

Additionally, a botulinum toxin may have an effect to reduce inducedinflammatory pain in a rat formalin model. Aoki K., et al, Mechanisms ofthe antinociceptive effect of subcutaneous BOTOX®: Inhibition ofperipheral and central nociceptive processing, Cephalalgia 2003September; 23(7):649. Furthermore, it has been reported that botulinumtoxin nerve blockage can cause a reduction of epidermal thickness. Li Y,et al., Sensory and motor denervation influences epidermal thickness inrat foot glabrous skin, Exp Neurol 1997; 147:452-462 (see page 459).Finally, it is known to administer a botulinum toxin to the foot totreat excessive foot sweating (Katsambas A., et al., Cutaneous diseasesof the foot: Unapproved treatments, Clin Dermatol 2002November-December; 20(6):689-699; Sevim, S., et al., Botulinum toxin-Atherapy for palmar and plantar hyperhidrosis, Acta Neurol Belg 2002December; 102(4):167-70), spastic toes (Suputtitada, A., Local botulinumtoxin type A injections in the treatment of spastic toes, Am J Phys MedRehabil 2002 October; 81(10):770-5), idiopathic toe walking (Tacks, L.,et al., Idiopathic toe walking: Treatment with botulinum toxin Ainjection, Dev Med Child Neurol 2002; 44(Suppl 91):6), and foot dystonia(Rogers J., et al., Injections of botulinum toxin A in foot dystonia,Neurology 1993 April; 43(4 Suppl 2)). Tetanus toxin, as wells asderivatives (i.e. with a non-native targeting moiety), fragments,hybrids and chimeras thereof can also have therapeutic utility. Thetetanus toxin bears many similarities to the botulinum toxins. Thus,both the tetanus toxin and the botulinum toxins are polypeptides made byclosely related species of Clostridium (Clostridium tetani andClostridium botulinum, respectively).

Additionally, both the tetanus toxin and the botulinum toxins aredichain proteins composed of a light chain (molecular weight about 50kD) covalently bound by a single disulfide bond to a heavy chain(molecular weight about 100 kD). Hence, the molecular weight of tetanustoxin and of each of the seven botulinum toxins (non-complexed) is about150 kD. Furthermore, for both the tetanus toxin and the botulinumtoxins, the light chain bears the domain which exhibits intracellularbiological (protease) activity, while the heavy chain comprises thereceptor binding (immunogenic) and cell membrane translocationaldomains.

Additionally, both the tetanus toxin and the botulinum toxins exhibit ahigh, specific affinity for gangliocide receptors on the surface ofpresynaptic cholinergic neurons. Receptor mediated endocytosis oftetanus toxin by peripheral cholinergic neurons results in retrogradeaxonal transport, blocking of the release of inhibitoryneurotransmitters from central synapses and a spastic paralysis.Contrarily, receptor mediated endocytosis of botulinum toxin byperipheral cholinergic neurons results in little if any retrogradetransport, inhibition of acetylcholine exocytosis from the intoxicatedperipheral motor neurons and a flaccid paralysis.

Finally, the tetanus toxin and the botulinum toxins resemble each otherin both biosynthesis and molecular architecture. Thus, there is anoverall 34% identity between the protein sequences of tetanus toxin andbotulinum toxin type A, and a sequence identity as high as 62% for somefunctional domains. Binz T. et al. The Complete Sequence of BotulinumNeurotoxin Type A and Comparison with Other Clostridial Neurotoxins, JBiological Chemistry 265(16); 9153-9158:1990.

Acetylcholine

Typically only a single type of small molecule neurotransmitter isreleased by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic as most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephine. In most instances acetylcholine has an excitatory effect.However, acetylcholine is known to have inhibitory effects at some ofthe peripheral parasympathetic nerve endings, such as inhibition ofheart rate by the vagal nerve.

The efferent signals of the autonomic nervous system are transmitted tothe body through either the sympathetic nervous system or theparasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

Acetylcholine activates two types of receptors, muscarinic and nicotinicreceptors. The muscarinic receptors are found in all effector cellsstimulated by the postganglionic neurons of the parasympathetic nervoussystem, as well as in those stimulated by the postganglionic cholinergicneurons of the sympathetic nervous system. The nicotinic receptors arefound in the synapses between the preganglionic and postganglionicneurons of both the sympathetic and parasympathetic. The nicotinicreceptors are also present in many membranes of skeletal muscle fibersat the neuromuscular junction.

Acetylcholine is released from cholinergic neurons when small, clear,intracellular vesicles fuse with the presynaptic neuronal cell membrane.A wide variety of non-neuronal secretory cells, such as, adrenal medulla(as well as the PC12 cell line) and pancreatic islet cells releasecatecholamines and parathyroid hormone, respectively, from largedense-core vesicles. The PC12 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,permeabilized (as by electroporation) or by direct injection of thetoxin into the denervated cell. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

A variety of substances, termed proteolytic enzymes, degrade or digestsubstances found in the extracellular matrix. These include the familyof hyaluronidases, plasminogen activators and collagenase, for example.Hyaluronidase causes hydrolysis of hyaluronic acid, a polysaccharide(nonsulfated glycosaminoglycan) found in the intercellular matrix ofconnective tissue. Hyaluronidase temporarily reduces the viscosity ofthe extracellular matrix (tissue cement) by digesting hyaluronic acid orhyaluronate, which is widely distributed throughout connective,epithelial, and neural tissues. This effect promotes the diffusion orspread of other drugs like anesthetic agents. Hyaluronidase may beinjected into connective tissue to enhance the effects of co-injecteddrugs.

Hyaluroronidase can be obtained from a variety of sources and istypically derived from testicular tissue extracts. For example, ISTAPharmaceuticals of Irvine, Calif., USA manufactures and distributesVITRASE (a sheep sourced (ovine) form of hyaluronidase), which is justone example of a hyaluronidase for injection. VITRASE is an injectabledrug approved by the U.S. FDA as an adjunct to (in combination with)other injected drugs to increase their absorption and dispersion. Asstated previously, hyaluronidase has been used most commonly incombination with local anesthetics in the setting of ophthalmic (eye)surgery. Hyaluronidase increases tissue permeability and promotes thespread or dispersion of other drugs, for example, speeding the onset ofaction for an anesthetic. VITRASE is also approved for use as an adjunctto rehydrating agents, and for use with certain imaging agents.Hyaluronidase is also available as a recombinant purified preparation ofthe enzyme recombinant human hyaluronidase, an example of which isHYLENEX, which is marketed by Baxter Healthcare Corporation, Deerfield,Ill., USA. HYLENEX (a recombinant hyaluronidase) is available as asterile clear, colorless, nonpreserved ready for use solution (each mLcontaining 150 USP units of recombinant human hyaluronidase with 8.f mgsodium chloride, 1.4 mg bibasic sodium phosphate, 1.0 mg human albumin,0.9 mg edetate, 0.3 mg calcium chloride, and sodium hydroxide for pHadjustment. Another exemplary hyaluronidase produced from sheep testesis named HYALASE, by Aventis Pharma, Lane Cove, NSW, Australia.

Hyaluronidase increases dispersion in the interstitial matrix providedlocal pressure is adequate to furnish the necessary mechanical impulse.Such an impulse is normally initiated by injected solutions and the rateof diffusion is proportionate to the amount of enzyme. The extent ofdiffusion is also proportionate to the volume of solution, as known inthe art.

Investigation of maintenance of efficacy, spread of effect and decreasein required dose of botulinum toxin administered along withhyaluronidase for treating axillary hyperhidrosis has been reported(“Diffusion and short-term efficacy of botulinum toxin A after theaddition of hyaluronidase and its possible application for the treatmentof axillary hyperhidrosis” by Goodman G. Dermatol Surg 2003 May;29(5):533-8. Here a formulation/mixture containing a botulinum toxin anda hyaluronidase is injected to treat hyperhidrosis, as well asadministration of botulinum toxin and superadded hyaluronidase.

Other proteolytic enzymes include collagenase and plasminogen activatorswhich digest extracellular matrix proteins. Plasminogen activators (PA)belong to a class of serine proteases that have considerable substratespecificity and convert the inactive zymogen plasminogen to plasmin.Plasmin is a general protease which is capable of degrading manyproteins including laminin, fibronectin and activating latentcollagenase moieties.

What is needed therefore is a method for treating various disorders thatreduces the amount of botulinum toxin administered to a patient. Moreparticularly a method is needed that reduces, or more preferably eveneliminates, the number of, or need for, injection of neurotoxins, suchas botulinum toxins, to treat various disorders.

SUMMARY

The present disclosure meets the need for a method by whichcholinerically-influenced disorders can be treated by reducing or eveneliminating the number of, and even the need for, injections endured bya patient in order to treat the disorder that the patient suffers from.

DEFINITIONS

As used herein, the words or terms set forth below have the followingdefinitions.

“About” means that the item, parameter or term so qualified encompassesa range of plus or minus ten percent above and below the value of thestated item, parameter or term.

“Target” or “target area” means that location/area or tissue or gland ofa patient's anatomy in which the desired effect of the administeredneurotoxin is exerted. A target can include, but is not limited to amuscle, such as a detrusor muscle of a bladder, or neurons thatinnervate a gland or muscle that is overactive, such as the neurons thatcontrol sweat production of sweat glands in the dermis of patient havinghyperhidrosis, or contraction of a targeted muscle, such a detrusormuscle and/or a urethral sphincter, for example. Typically, the targetis within 5 inches of the locale of the administration of a compositionof the instant invention, preferably within 3 inches and even morepreferably within 1 inch.

“Administration”, “administering” or “to administer” means the step ofgiving (i.e. administering) a composition to a subject, such as apharmaceutical composition. The pharmaceutical compositions disclosedherein are “locally administered” by e.g. intramuscular (i.m.),intradermal, subcutaneous administration, intraperitoneal (i.p.)administration, topical (transdermal), instillation (e.g. intravesicularinstillation) and implantation (e.g. a slow-release device such aspolymeric implant) routes of administration.

“Botulinum toxin” means a neurotoxin produced by Clostridium botulinum,as well as a botulinum toxin (or the light chain or the heavy chainthereof) made recombinantly by a non-Clostridial species. The phrase“botulinum toxin”, as used herein, encompasses the botulinum toxinserotypes A, B, C₁, D, E, F and G. Botulinum toxin, as used herein, alsoencompasses both a botulinum toxin complex (i.e. the 300, 600 and 900kDa complexes) as well as the purified botulinum toxin (i.e. about 150kDa). “Purified botulinum toxin” is defined as a botulinum toxin that isisolated, or substantially isolated, from other proteins, includingproteins that form a botulinum toxin complex. A purified botulinum toxinmay be greater than 95% pure, and preferably is greater than 99% pure.The botulinum C₂ and C₃ cytotoxins, not being neurotoxins, are excludedfrom the scope of the present invention.

“Clostridial neurotoxin” means a neurotoxin produced from, or native to,a Clostridial bacterium, such as Clostridium botulinum, Clostridiumbutyricum or Clostridium beratti, as well as a Clostridial neurotoxinmade recombinantly by a non-Clostridial species.

“Modified botulinum toxin” means a botulinum toxin that has had at leastone of its amino acids deleted, modified, or replaced, as compared to anative botulinum toxin. Additionally, the modified botulinum toxin canbe a recombinantly produced neurotoxin, or a derivative or fragment of arecombinantly made neurotoxin. A modified botulinum toxin retains atleast one biological activity of the native botulinum toxin, such as,the ability to bind to a botulinum toxin receptor, or the ability toinhibit neurotransmitter release from a neuron. One example of amodified botulinum toxin is a botulinum toxin that has a light chainfrom one botulinum toxin serotype (such as serotype A), and a heavychain from a different botulinum toxin serotype (such as serotype B).Another example of a modified botulinum toxin is a botulinum toxincoupled to a neurotransmitter, such as substance P.

A “therapeutically effective” amount of the neurotoxin is the dosagesufficient to inhibit neuronal activity for at least one week, morepreferably one month, most preferably for approximately 6 to 9 months orlonger and up to 5 years. Dosing can be single dosage or cumulative(serial dosing), and can be readily determined by one skilled in theart. Neurotoxin, such a botulinum toxin, can be delivered serially(i.e., one time per month, one time per every six months) such that anoptimal amount of toxin is administered in accordance with the severityof the disorder treated and beneficial results are maintained. Such adosage schedule is readily determined by one skilled in the art basedon, e.g., patient size, the neurotoxin selected, the condition to betreated, severity of the disorder and other variables known in the art.

“Patient” means a human or non-human subject receiving medical orveterinary care. Accordingly, as disclosed herein, the compositions maybe used in treating any animal, such as mammals.

“Sufficient amount” means that amount of a substance, composition ofelement of a composition that is enough to meet the needs under thesituation or a proposed end. For example, in treating a particulardisorder, it is that amount of botulinum toxin that results in a desiredoutcome, e.g. a decrease in detrusor muscle spasm or decrease inexcessive sweat production.

“Cholinergically-influenced disorder” is a disorder that results fromthe dysfunction of a gland, organ or tissue that is the result of overor under activity of the gland, organ or tissue, or abnormal/disruptiveenlargement of the gland organ or tissue, wherein the gland organ oftissue is influenced/innervated by acetylcholine releasing neurons.Non-limiting examples of cholinergically-influenced disorders include,hyperhidrosis, overactive bladder, and benign prostatic hyperplasia, forexample. The term “urologic disorder” includes, but is not limited to,overactive bladder, detrusor hyperreflexia, detrusor instability,neurogenic bladder, idiopathic bladder, benign prostate hyperplasia andurinary incontinence.

An extracellular matrix digesting enzyme is an enzyme thatdigests/breaks down at least one component of the extracellular matrix.Exemplary extracellular matrix digesting enzymes include hyaluronidase,which digests hyaluronic acid and has potential application in both thebladder and prostate for disorders such as overactive bladder,neurogenic bladder, benign prostatic hyperplasia, prostitis, andprostate cancer. Other enzymes which digest the extracelluar matrixincluding collagenase and plasminogen activators such as tissueplasminogen activator and urokinase which may have similar applicationby digesting the extracellular matrix to thereby enhancing diffusion ofneurotoxins, and reduce the number of or eliminate the need forinjection of neurotoxin, in accordance with one aspect of the presentteachings.

A surface area is simply a particular area on the surface of asubject/patient, such as a skin surface, to which compositions of theinstant disclosure are administered. Non-limiting examples of a surfacearea include an axillary skin surface area, a palmar skin surface areaand a plantar skin surface area.

A “luminal surface area” of a patient/subject is an area that faces alumen, as well known in the art. Non-limiting examples include a bladderluminal surface area, nasal luminal surface area, a prostate luminalsurface area, an esophageal luminal surface area, stomach luminalsurface area, intestinal luminal surface area and a vascular luminalsurface area, for example.

“Intravesically administered” or “intravesical administration” meansinstillation of a composition into a lumen to contact a luminal surfacearea, such as a bladder luminal surface area, for example, by any knownsuitable and appropriate means. Intravesical administration excludes,however, injection into a wall facing the lumen, such as a bladder wall.

“Alleviating” means a reduction in the occurrence of a symptom that isassociated with a cholinergically-influenced disorder. For example,alleviating includes some reduction, significant reduction, near totalreduction, and total reduction of at least one symptom associated withhyperhidrosis, overactive bladder, and benign prostate hyperplasia, forexample, or any disorder treated in accordance with the methodsdisclosed herein. An exemplary symptom of hyperhidrosis is excessivesweating, for overactive bladder and benign prostate hyperplasia,exemplary symptoms can be incontinence or retention, for example. Analleviating effect may not appear clinically for between 1 to 7 daysafter administration of a Clostridial toxin, such as a botulinum toxin,to a patient.

“Treating” means to alleviate (or to eliminate) at least one symptom ofa cholinergically-influenced disorder, either temporarily orpermanently.

A method for treating a patient having a cholinergically-influenceddisorder, in accordance with the present disclosure, can comprise thesteps of administering a first composition containing an extracellularmatrix digesting enzyme to a surface area of the patient, followed byallowing a sufficient amount of time to pass for the extracellularmatrix digesting enzyme to diffuse through the surface area, thenadministering a second composition containing a botulinum toxin to thesurface area, and subsequently allowing sufficient time for thebotulinum toxin to diffuse through the surface area to thereby alleviateat least one symptom associated with the cholinergically-influenceddisorder and treat the patient having the cholinergically-influenceddisorder. In particular instances, the surface area is a luminal surfacearea such as a bladder luminal surface area. The extracellular matrixdigesting enzyme is a hyaluronidase, tissue plasminogen activator andcollagenase, for example, while the botulinum toxin is selected from thegroup consisting of botulinum toxin type A, B, C, D, E, F, and G.

Various methods of administration can be utilized for administration ofthe compositions useful in practicing the methods disclosed herein. Inone instance, administration of the extracellular matrix digestingenzyme and botulinum toxin to a bladder luminal surface area is achievedby instillation of a composition containing an extracellular matrixdigesting enzyme, as well as instillation of a composition containingbotulinum toxin, into a bladder, for example.

Additionally, due to the synergistic effects provided by methodspracticed in accordance with the teachings disclosed herein,administration of the extracellular matrix digesting enzyme can beaccomplished by injection, for example into a bladder wall, orsubdermally injected to a skin surface area (such as into an armpit(axilla), palmer or plantar surface, for example), while administrationof a neurotoxin containing second composition is accomplished byinstillation into the bladder, or sprayed, swabbed or smeared onto theskin surface area, respectively, thereby avoiding any need for injectionof the botulinum toxin. Conversely, it is also contemplated thatadministration of the extracellular matrix digesting enzyme (a firstcomposition) can be accomplished by instillation of the firstcomposition into a bladder or sprayed, swabbed or smeared onto the skinsurface area, and administration of the botulinum toxin is accomplishedby injection of the second composition (containing a neurotoxin, such asa botulinum toxin) into a bladder wall or subdermally into the skinsurface area, respectively.

Accordingly, administration of the botulinum toxin can be achieved byless than 20 injections into the bladder wall, more preferably by lessthan 10 injections into the bladder wall and most preferably byperforming between 1 and 5 injections into the bladder wall. Forexample, a total of 5 injections of neurotoxin, such as botulinum toxin,after administration of the first composition having the extracellularmatrix digesting enzyme, can be administered as follows: 1 injection tothe dome of a bladder, 1 injection to an ventral wall of the bladderwall, 1 injection to a dorsal wall of the bladder, and 1 injection eachinto each lateral wall (left and right lateral wall of the bladder) fora total of five injections. Particularly useful botulinum toxin includebotulinum toxin selected from the group consisting of botulinum toxintypes A, B, C₁, D, E, F and G.

In particular embodiments, the methods disclosed herein can includefurther steps of emptying the bladder prior to administration of acomposition containing an extracellular matrix digesting enzyme andoptionally emptying the bladder after administration of the compositioncontaining the extracellular matrix digesting enzyme, and optionallyemptying the bladder after administration of the second composition thatcontains a neurotoxin, such as a botulinum toxin. As stated above, themethods disclosed herein can include removing the first composition(containing at least one extracellular matrix digesting enzyme) andremoving the second composition (containing at least one neurotoxin,such as a botulinum toxin).

Exemplary cholinergically-influenced disorders that can be treated inaccordance with the instant disclosure include a urologic disorder suchas a bladder disorder or a prostate disorder. Exemplary bladderdisorders include overactive bladder, hypertrophied bladder neck anddetrusor hyperreflexia, for example. Exemplary prostate disordersinclude benign prostatic hyperplasia, prostatitis and prostate cancer.An additional example of a cholinergically-influenced disorder ishyperhidrosis and the surface area of the patient, when treatedaccordingly, can is selected from the group consisting of an axillaryskin surface area, a palmar skin surface area and a plantar skin surfacearea.

In particular embodiments, in addition to reducing the number ofinjections utilized to treat cholinergically-influenced disorders, themethod for administering a neurotoxin to a patient in need thereof canspecifically exclude any injection of the neurotoxin or an extracellularmatrix digesting enzyme, where the method comprises the steps ofadministering a first composition containing at least one extracellularmatrix digesting enzyme onto a skin surface area or luminal surface areaof the patient and administering the second composition containing aneurotoxin onto the skin surface area or luminal surface area of thepatient, where the neurotoxin diffuses to a greater extent that ifadministered without the first composition containing at least oneextracellular matrix digesting enzyme, and further the administrationexcludes injection of both the first and second compositions. In suchembodiments, the skin surface area can be an axillary skin surface area,plantar skin surface area or palmar skin surface area. An exemplaryluminal surface area can be a bladder luminal surface area, a urethralluminal surface area, a nasal luminal surface area or a prostate luminalsurface area.

For example, in methods that specifically exclude injection of theneurotoxin or an extracellular matrix digesting enzyme, theadministration of one or both of the extracellular matrix digestingenzyme and botulinum toxin is achieved by application via at least oneof spraying or rubbing onto the skin surface area or luminal surfacearea of the patient. A method can further include the step of drying theskin surface after administration of the first composition (containingat least one extracellular matrix digesting enzyme) to the skin surface.Drying the skin surface can include the step of allowing sufficient timeto pass to allow evaporation of the first composition from the skinsurface, before commencing with administration of the second composition(containing a neurotoxin, such as a botulinum toxin selected from thegroup consisting of botulinum toxin type A, B, C₁, D, E, F or G), ontothe skin surface area.

Another non-injection method is provided in accordance with the instantdisclosure, for treating a urologic disorder in a patient in needthereof, comprising the steps of instilling a first compositioncontaining hyaluronidase into to a bladder of the patient in order tocontact a bladder luminal surface area (which has a glycosaminoglycanlayer) to the first composition and maintaining the first compositionwithin the bladder to allow sufficient time to pass such that theintroduced (and instilled) hyaluronidase interacts with theglycosaminoglycan layer and diffuses through the bladder luminal surfacearea, optionally draining the first composition from the bladder,instilling a second composition containing a botulinum toxin type A tothe bladder in order to contact the bladder luminal surface areapreviously contacted by the previously instilled and removed firstcomposition, and retaining the instilled second composition for asufficient time within the bladder so that a sufficient amount ofbotulinum toxin type A diffuses through the bladder luminal surface areato at least one layer of the muscularis propria (at least one of theinner longitudinal, middle circular, and outer longitudinal layers) andoptionally draining the second composition from the bladder, therebyalleviating at least one symptom associated with the urologic disorderand treating the urologic disorder of the patient in need thereof. It isfurther contemplated that a single composition/mixture that includesboth an extracellular matrix digesting enzyme and a botulinum toxintherein can be instilled into a bladder.

Exemplary urologic disorders that can be so treated, that is, by methodsthat do not require the use of injections, include urologic disordersselected from the group consisting of overactive bladder, hypertrophiedbladder neck and detrusor hyperreflexia, for example.

Each and every feature described herein, and each and every combinationof two or more of such features, is included within the scope of thepresent invention provided that the features included in such acombination are not mutually inconsistent.

DESCRIPTION

The present disclosure provides methods by which variouscholinergically-influenced disorders, such as urologic disorders andhyperhidrosis can be treated. Urologic disorders include overactivebladder, hypertrophied bladder neck and detrusor hyperreflexia, forexample, which can be treated by utilizing an extracellular matrixdigesting enzyme in conjunction with a neurotoxin, such as botulinumtoxin as taught herein. Such use enhances the diffusion of botulinumtoxin and as such can reduce, and even eliminate, the need for injectionprotocols that are typically utilized when treated these disorders withbotulinum toxin.

In accordance with the present disclosure, the methods are describedherein that take advantage of the synergistic effect of utilizing atleast one extracellular matrix digesting enzyme in conjunction with aneurotoxin, preferably a botulinum toxin, in order to treat variousdisorders, as more fully described below. An advantageous aspect of themethods detailed herein is the reduction in the number of, and evenelimination of, injections to administer therapeutically effectiveamounts of the neurotoxin to the patient and thereby treat the disorder.

The neurotoxin can be formulated in any pharmaceutically acceptableformulation/formulations such as a liquid, powder, cream, emulsion,suspensions, solutions, and the like.

The amount of the Clostridial toxin, such as botulinum toxinadministered according to a method within the scope of the disclosedherein can vary according to the particular characteristics of thedisorder being treated, for example, such a urologic disorder orhyperhidrosis, including the severity and other various patientvariables including size, weight, age, and responsiveness to therapy, asknown in the art. To guide the practitioner, typically, no less thanabout 1 unit and no more than about 2500 units of a botulinum toxin typeA (such as BOTOX®) is administered per injection site if the toxin isinjected, per patient treatment session. For a botulinum toxin type Asuch as DYSPORT®, no less than about 2 units and no more about 4000units of the botulinum toxin type A are administered per injection site,per patient treatment session, if injected. For a botulinum toxin type Bsuch as MYOBLOC®, no less than about 40 units and no more about 25,000units of the botulinum toxin type B are administered per injection site,per patent treatment session. Similar amounts of toxin can be utilizedin accordance with methods that do not utilize injection of toxin, suchas instillation, swabbing or spraying of neurotoxin containingcompositions to areas to which a composition containing an extracellularmatrix digesting enzyme has been or is/will be administered, either byinjection or non-injection. Of course, the amount of neurotoxin andextracellular matrix containing enzyme to be utilized in a particularpatient to treat a particular disorder/condition will be determined bythe attending physician, as known in the medical arts. For example, whentreating a urologic disorder by administration of neurotoxin to apatient's bladder, the volume of the solution/dispersion andconcentration of the neurotoxin may depend upon the size of the patient,the severity of the disorder, thickness of the bladder wall,concentration/amount of administered extracellular matrix digestingenzyme and muscle, comorbidities, and other factors.

For example, if treating a patient suffering from hyperhidrosis, such asaxillary hyperhidrosis, the hyperhidrotic surface area to be treated(here an armpit) is first determined by conducting a simple Minor'sstarch and iodine test, in order to determine the area to be treated.The area is demarcated, and a composition containing an extracellularmatrix digesting enzyme, such as a solution containing hyaluronidase, isapplied to the hyperhydrotic area. For example, the composition soapplied can contain about 150 U of hyaluronidase and is left on thepatient's skin surface to allow the hyaluronidase to be absorbed. Suchapplication can be simply accomplished by brushing, spraying or swabbinga first composition containing an extracellular matrix digesting enzymeonto the desired area. In some instances, sufficient time is allowed topass to allow the skin surface area to dry. Subsequently, a compositionthat contains a neurotoxin, such as a botulinum toxin, is applied to thearea. Such a composition can contain, for example, from about 50 toabout 200 units of a botulinum toxin. Preferably, the compositioncontaining the botulinum toxin is similarly brushed, sprayed or swabbedto the surface area to be treated and the patient can report a decreasein excessive sweating and a return to euhidrosis in about 2 to about 7days time.

Alternatively, for example, the botulinum toxin can be administered byinjection, such as by subdermal injection. However, due to the topicalapplication of the extracellular matrix digesting enzyme, such ashyaluronidase, the number of necessary injections is greatly reduced ascompared to the typical number of injections of botulinum toxin utilizedto treat axillary hyperhidrosis. As an example, between about 5 to about10 injection sites, having between about 5 to about 25 units of BOTOX®(botulinum toxin type A) at each site, can be administered to the areato which the extracellular matrix digesting enzyme is administered.Preferably, up to about 5 injections of botulinum toxin are administeredto the area to which the extracellular matrix digesting enzyme isadministered. Typically, the injections are evenly spaced from oneanother to as to cover the maximum amount of hyperhidrotic area.

In another example, the methods disclosed herein provide for methods totreat various urological disorders, for example, by administering aneurotoxin such as a botulinum toxin to a bladder's luminal surface areato which has been administered a composition that contains anextracellular matrix digesting enzyme. Access to the lumen of thebladder is easily accomplished by insertion of a catheter or cannulainto the urethra and to the bladder, as known in the art. Once thecatheter is so positioned, between 1 and 1000 ml of a first compositioncontaining extracellular matrix digesting enzyme, such as hyaluronidase,is instilled into the bladder. The solution can contain anywhere fromabout 25 to about 50 Units of hyaluronidase, the volume of thecomposition (for example a solution) and concentration of thehyaluronidase may depend upon the size of the patient, thickness of thebladder wall and muscle, comorbidities, severity of the urologicdisorder, weight of the patient among other standard factors consideredin the medical arts when determining appropriate dosages/parameters fortreating particular patients. The first composition may then be drainedfrom the bladder after allowing a sufficient amount of time to pass,such as from about 5 minutes to about 2 hours. In some embodiments,where, for example, from about 10 ml to about 50 ml of the firstcomposition is instilled into the bladder, there may not be a need todrain the bladder, as the composition can be absorbed. During this time,the patient may be positioned (turned on their sides, onto their stomachand back) in order to thoroughly establish contact of the firstcomposition with the bladder luminal surface. The first composition canthen be drained from the bladder (utilizing known drainage techniques,and can include external, manual depression of the bladder, forexample). Subsequently, a second composition containing a neurotoxin,preferably a botulinum toxin, most preferably a botulinum toxin type A,is then administered by instillation into the bladder to contact thebladder luminal surface previously administered the first compositioncontaining the extracellular matrix digesting enzyme. From about 25 toabout 3000 units, more preferably from about 100 to about 2500 units ofa botulinum toxin type A can be so instilled, and from about 500 toabout 50,000 units, and more preferably from about 1000 to about 25,000units of a botulinum toxin type B can be so instilled into the bladderor a clinically equivalent amount for other botulinum toxin serotypes,as known to the skilled person in the art.

The dosage of neurotoxin agent that is intravesically administered tothe patient is one that is therapeutically effective to achieve thedesired treatment outcome. In the case of botulinum toxin, the typicaldose administered to the patient may be any dose less than a toxic dose(for example less than 3000 units of BOTOX®, a botulinum toxin type A,for a 70 kg man), for example between 1 and 1,500 units and morepreferably between 50 and 500 units per patient per treatment, althoughsmaller or larger doses may be administered as required. The doses canbe given as a single dose, or as divided doses over a span of time, suchas over a period of days or weeks or months, depending on the length ofeffect for a given neurotoxin preparation.

Similar to the first composition, between about 1 and about 1000 ml ofthe second composition containing botulinum toxin can be instilled intothe bladder and the patient placed in various positions as detailedabove. Because the patient will likely be instructed to empty his or herbladder prior to the procedure, the bladder will likely not be full ormarkedly distended. In particular embodiments, instillation of about 1to about 100 mls of solution/dispersion, and more preferably 10-50 ml ofsolution/dispersion, may be sufficient to coat the inside of the bladder(bladder luminal surface). Additionally, after a sufficient amount oftime (e.g. from about 5 minutes to about 2 hours) has passed afterinstillation of the second composition into the bladder, the secondcomposition containing the botulinum toxin can be drained from thebladder, although if a smaller volume of the second composition isinstilled (e.g. from about 1 to about 10 mls), the attending physicianmay not desire to drain the bladder and rather allow for the secondcomposition to be naturally drained (expelled) by the patient. Acomposition (containing either/or a botulinum toxin or an extracellularmatrix digesting enzyme) for bladder infusion according to the teachingof the present disclosure typically is of a volume of about 80 to about100 ml, and more preferably 80 ml. Of course, the attending physiciancan increase or decrease the concentration of the neurotoxin containingcomposition and extracellular matrix digesting enzyme composition, andvolume of the instilled compositions, in accordance with the patient'sbladder size (children and young adults having smaller bladders thanadults) and severity of the disorder treated.

Draining of the instilled compositions can be accomplished via catheteror naturally expelled, appropriate care being taken, of course,associated with the disposable of neurotoxin containing compositions.Within about 2 to about 7 days the patient can report urologicalimprovement and even a return towards a normal urological state, which,for an adult, is having a flow rate of about 25 cc/sec and a void volumeof about 400 cc.

Draining of the first composition (containing the extracellular matrixdigesting enzyme) and the second composition are described above forinstillation into a bladder. If instilled into a portion of a patient'sGI tract or into a nasal lumen, appropriate routes of removal/drainagecan be employed, such as simply tilting/positioning the patients head toinstill or remove compositions from a nasal luminal surface area, forexample.

An exemplary method for intravesically administering the first andsecond compositions utilizes a urinary catheter that extends through theurethra into the bladder. The catheter may be a “straight catheter” witha single lumen (simply a straight channel) or alternatively might be acatheter that in some cases uses a balloon or other mechanism to fix thecatheter within the bladder (such as a Foley catheter). Standard sizesfor such a catheters are known in the art, such as 10-16 French (3-5mm), though larger or smaller sizes might be used depending on size thepatient and his or her anatomy.

Once the catheter is in place, typically between 1 and 1000 ml ofsolution/dispersion containing neurotoxin and more preferably in therange of 10-50 ml of solution/dispersion containing neurotoxin can beinstilled through the catheter into the bladder. The volume of thesolution/dispersion and concentration of the neurotoxin may depend uponthe size of the patient, thickness of the bladder wall and muscle,comorbidities, and other factors.

Another representative means of intravesically administering theneurotoxin involves the placement of a suprapubic needle or catheterthrough the abdominal wall directly into the patient's bladder. This isa more invasive method and is not the preferred method of access to thebladder; however, due to urethral tract infections, obstructions, etc.,may be the best route that is available to the attending physician toaccess the bladder luminal surface. The required volume of compositionscontaining the extracellular matrix digesting enzyme and the neurotoxincan then be introduced into the bladder, either using direct vision,endoscopic or fluoroscopic guidance, as known in the art. Intravesicaladministration in accordance with the present disclosure can also beaccomplished utilizing a cystoscope which facilitates viewing ofintravesical delivery of the compositions. Here, the compositions can beintroduced into the bladder lumen through the working channel of thecystoscope or through a catheter or other tubular structure passedwithin or alongside the cystoscope.

In some cases, the urethra or suprapubic catheter/needle may have aninflatable component that can be inflated within the bladder to “lock”the urethra or suprapubic catheter/needle in place and prevent itsremoval. Inflating the balloon or other inflatable device takes upvolume within the bladder, and can thereby require less of theextracellular matrix digesting enzyme composition and neurotoxincontaining composition to be administered.

In accordance with once aspect, the extracellular matrix digestingenzyme and a botulinum toxin can be serially administered oradministered at the same time. An exemplary mixture for instillation orspreading to a surface area in can, for, example, containing 100 unitsof botulinum toxin type A (BOTOX) diluted with 9 ml of preserved salineand 1 ml of hyaluronidase (1500 units, here HYALASE).

Although examples of routes of administration and dosages are provided,the appropriate route of administration and dosage are generallydetermined on a case by case basis by the attending physician. Suchdeterminations are routine to one of ordinary skill in the art (see forexample, Harrison's Principles of Internal Medicine (1998), edited byAnthony Fauci et al., 14th edition, and published by McGraw Hill). Forexample, the route and dosage for administration of a Clostridialneurotoxin, such as a botulinum toxin, according to the presentdisclosed invention can be selected based upon criteria such as thesolubility characteristics of the neurotoxin chosen as well as theintensity of the disorder treated.

The following examples provide those of ordinary skill in the art withspecific preferred methods to practice methods that are within the scopeof the present invention and are not intended to limit the scope of theinvention.

EXAMPLES Example 1

A 64-year old patient has an overactive bladder, and as a result hasurge and stress incontinence. He unfortunately experiences from about5-8 leakage accidents per day, requiring necessary changes of the adultdiapers that he is forced to wear because of his condition. Uponpresentation to his urologist, a regimen of bladder instillation isdecided upon, utilizing botulinum toxin and an extracellular matrixdigesting enzyme.

The patient is asked to relieve himself before lying on his back upon anadjustable table or bed, after which a urethral catheter is insertedinto his urethra and to the patient's bladder. A first compositioncontaining an extracellular matrix digesting enzyme, here 150 USP unitsof nonpreserved hyaluronidase (such as HYLENEX) in 50 mls ofnonpreserved saline, are instilled into the man's bladder. The catheteris then removed and the surface upon which the patient is lying istilted so that his head is lower than his feet, in order that the firstcomposition contacts the bladder luminal surface at the dome of thebladder. The patient remains in such a position for 10 minutes, afterwhich he is titled forward so that his feet are lower than his head sothat the first composition is now in full contact with the floor of thebladder. The patient remains so for 10 more minutes, and is then askedto roll onto his left and right sides (for 10 minutes each,respectively) and then onto his stomach, to more fully expose all of thebladder's lateral luminal surface areas to the first composition thatincludes the an extracellular matrix digesting enzyme. The patient isthen recatheterized and stood upright, to drain the bladder. Then asecond composition, which contains 500 units of a botulinum toxin typeA, such as BOTOX® reconstituted in 50 mls of non-preserved saline, isthen instilled into the patient's bladder and the patient is thensubjected to the same positioning regimen as for the first composition.Subsequently, the patient is drained of the second composition anddischarged.

Weekly follow up visits show that the patient now has control over hisurination, and although he still wears adult diapers out of abundance ofcaution, and does not have accidental leakage episodes since theinstillation treatment and can enjoy running and other physicalactivities that his stress incontinence forces him to avoid.

Example 2

A 72-year old man has suffers from urge incontinence due to a neurogenicbladder dysfunction that is secondary to his Parkinson's disease. Hiscondition forces the patient to make, on average, over 20 trips to therestroom per day to relieve his bladder. The situation is presented tohis physician and administration of an extracellular matrix digestingenzyme and botulinum toxin to his bladder walls is decided upon.

The patient is firstly asked to relive himself before insertion of anappropriately sized catheter (3-5 mm) into his urethra. A firstcomposition containing 300 USP units of nonpreserved hyaluronidase (suchas HYLENEX) in 2 mls of solution is instilled into the bladder and apositioning routine, however the patient now remains in the variouspositions for 5 minutes. After drainage of the patient's bladder, acytoscope is utilized to inject botulinum toxin at one site into each ofthe dome, dorsal, ventral and lateral walls of the bladder, sparing thetrigone. Thus a total of five injections, of about 10 units of abotulinum toxin type A complex at each injection site for a total ofabout 50 units of botulinum toxin type A (e.g. BOTOX®, or about 40 unitsof DYSPORT® at each site, for a total of 200 units of botulinum toxintype A) are so delivered, a great reduction from the typically 20-40injections of prior methods which utilizing toxin alone. Theinstillation of the extracellular matrix digesting enzyme into thebladder and to the bladder luminal surface facilitates the greaterdiffusion of the toxin throughout the muscularis propria (i.e. the threelayers: inner longitudinal, middle circular, and outer longitudinalmuscles of the bladder's muscular layer) while reducing the amount ofinjections that need to be performed.

The patient rests after the procedure is completed and is then takenhome. During follow-up visits, the patient reports no unwanted systemicor local side effects and shows an improvement in bladder function, bothsubjectively (reduction in urgency to urinate) and objectively (now onlyurinates 4 times/day on average).

Injection of up to 5000 units of botulinum toxin type B, at 1000 unitsper injection site can also be performed and treats neurogenic bladderdysfunction that is secondary to his Parkinson's disease, similarly.

Example 3

A 58 year old woman suffers urinary retention that is secondary tospastic sphincter and has a hypertrophied bladder neck. The patienttypically urinates only 2-3 times per week and when so doing onlymanages to void approximately 50 mls of urine per visit to the restroom.Her caretaker fears resultant high intravesical pressure and reductionin bladder capacity will result in deterioration of the upper urinarytract.

Accordingly, her urologist inserts a cytoscope and flexible endoscopicneedle into her urethra and into the bladder. A first compositioncomprising a total of 100 units of hyaluronidase, such as VITRASE, isinjected into the detrusor muscle, at a total of 10 sites, 2 sites intoeach into of the dorsal, ventral and lateral bladder walls (for a totalof 8 injections to the bladder walls) and 2 sites into the bladder neckand the cytoscope removed. After 10 minutes, a second composition of 100mls of nonpreserved saline, containing 750 units of a botulinum toxintype A (e.g. DYSPORT®) is instilled into bladder and kept there for 30minutes, after which the patent is recatherized and voided of the secondcomposition. Within 7 days, the caretaker reports improved frequency ofurination (1 to 2 times per day) and an increase in the amount of urinevoided (about 300 to 400 mls per visit to the restroom).

Example 4

A 23 year old college student reports to her dermatologist stating thather excessive axillary sweating is rendering her very self conscienceand as a result her grades are suffering and she has become more andmore introverted as time passes. The dermatologist determines that thepatient is suffering from hyperhidrosis and suggests utilizing aneurotoxin to treat the hyperhidrosis since roll-on antiperspirants haveno effect on her excessive sweating.

After wiping the axilla of the patient dry with a paper towel, a Minor'siodine-starch test is performed to demarcate the hyperhidrotic skinsurface area to be treated (such as by utilizing a vegetable-based ink).Applied to this axillary skin surface area is a first composition thatcontains an extracellular matrix digesting enzyme, here 1 ml containing150 USP units of nonpreserved hyaluronidase (such as HYLENEX) is appliedby swabbing the demarcated area with an applicator, such as a cottonswab. The area is then allowed to air-dry, for about 10 minutes.Thereafter and to the axillary skin surface area, about 25 units ofbotulinum toxin type A (such as BOTOX®, or about 100 units of DYSPORT®,or about 1250 units of MYOBLOC®) is applied, utilizing another cottonswab and allowed to dry. The same procedure is performed on thepatient's other axilla.

Within one week, the patient reports that they no longer experience theexcessive sweating that pervaded their previous days, and that there areno excessive local hypotonicity or systemic adverse effects.

A similar approach can be utilized to treat other skin surface areasthat may also be hyperhidrotic, such as the palms of the hands (palmarskin surface area) and/or the soles of the feet (plantar skin surfacearea).

Example 5

A 42 year old single man reports to his dermatologist that his excessiveaxillary sweating is rendering him very self conscience to the point ofno longer interacting with the opposite sex. The dermatologistdetermines that the patient is suffering from hyperhidrosis and suggestsutilizing a neurotoxin to treat the hyperhidrosis, since all of thetopical antiperspirants he as tried have not been effective.

After wiping the axilla of the patient dry with a paper towel, a Minor'siodine-starch test is performed to demarcate the hyperhidrotic skinsurface area to be treated (such as by utilizing a vegetable-based ink).Applied to this axillary skin surface area is a first composition thatcontains an extracellular matrix digesting enzyme, here 2 mls containing300 USP units of nonpreserved hyaluronidase (such as HYLENEX), appliedby spraying the demarcated area with a spray applicator, such as anon-aerosol pump. The area is then allowed to air-dry, for about 10minutes. Thereafter and to the axillary skin surface area, about 1000units of botulinum toxin type B (such as MYOBLOC®) is sprayed thereon,utilizing a spray applicator and allowed to dry. The same procedure isperformed on the patient's other axilla.

Within 10 days, the patient reports that he no longer experiences theexcessive sweating that pervaded their previous lonely days, and furtherthat there are no excessive local hypotonicity or systemic adverseeffects. The patient reports that the procedure was effective forbetween about 2 to about 6 months, at which time he returns to thedermatologist for another round of treatment.

Example 6

A 15 year old high school student reports to his family physician thathis hyperhidrotic hands are a source of great embarrassment.Accordingly, the doctor proceeds to perform a Minor's iodine-starch testto demarcate the areas to be treated. It appears that the excessivesweating mainly originates from the area between the patient's wrists tothe bases of the fingers.

Accordingly, 1 ml of hyaluronidase containing 150 USP units is appliedto the wrist to the base of the fingers (1 ml/150 USP units per hand)and allowed to dry. After drying, 4 points of injection in the palm(midline at the base of wrist, base of middle finger, and between baseof the thumb and base of the index finger, and between base of the pinkyand base of wrist) at which about 40 units of botulinum toxin type A(such as BOTOX®, or about 80 units DYSPORT® or 200 units of a botulinumtoxin type B, such as MYOBLOC®) is injected intradermally at each of thefour points.

After 5 days, the teen reports that his palms are not longer sweaty andthere are no reports of excessive hypotonicity or systemic effects. Thehyperhidrosis abates for up to 8 months, at which time the teen returnsto have the procedure repeated. If needed, the treatment is also appliedto the ventral portions of the patient's fingers, if exhibitingexcessive sweating.

Example 7

A 76 year old man suffers from chronic urinary retention due to theenlargement of his prostate due to benign prostatic hyperplasia. It isdecided by his physician that the patient undergo administration of abotulinum toxin to the prostate in order to alleviate his urinaryretention and treat the benign prostatic hyperplasia. In order tominimize the number of injections of botulinum toxin to the prostate, anextracellular matrix enzyme, such as hyaluronidase, is injected orinstilled into the transition zone of the lateral lobes of the prostateand the median lobe. Three injections of hyaluronidase (50 units atinjection point) are made utilizing an injection cytoscope and a 23gauge needle, one injection into each of the lateral lobes and oneinjection into the median lobe. After 5 minutes, three injections of abotulinum toxin type A are similarly administered, where each injectioncontains 50 units of botulinum toxin type A (BOTOX®) for a total of 150units.

After about 7 days, the patient reports an improvement in spontaneousvoiding after this treatment and decrease post voiding residual volumeand pressure are decreased. These beneficial effects are maintained forabout 5 months in this particular patient and no adverse effects arereported. A type B botulinum toxin can also be utilized, for example,from about 250 units to 1000 units per injection site.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe methods of the present invention in place of Clostridialneurotoxins. Additionally, the present invention includes administrationof two or more different Clostridial toxin components and targetingmoieties, administered concurrently or consecutively. For example,administration of a particular second composition containing botulinumtoxin type A to the bladder luminal surface or hyperhidrotic skinsurface area can be administered to the patient and if increasedtolerance resistance to it's effect is noted, a botulinum toxin of adifferent serotype, such as botulinum toxin type B or F, can be utilizedin subsequent applications to treat the cholinergically-influenceddisorder. While this invention has been described with respect tovarious specific examples and embodiments, it is to be understood thatthe invention is not limited thereto and that it can be variouslypracticed with the scope of the following claims.

1. A method for treating a patient having a cholinergically-influenceddisorder, comprising the steps of: a) administering a first compositioncontaining an extracellular matrix digesting enzyme to a surface area ofthe patient; b) allowing sufficient time to pass for the extracellularmatrix digesting enzyme to diffuse through the surface area and to atarget; d) administering a second composition containing a botulinumtoxin to the surface area; and e) allowing sufficient time for thebotulinum toxin to diffuse through the surface area and to the target,thereby alleviating at least one symptom associated with thecholinergically-influenced disorder and treating the patient having acholinergically-influenced disorder.
 2. The method according to claim 1,wherein the surface area is a luminal surface area.
 3. The method ofclaim 2, wherein the luminal surface area is a bladder luminal surfacearea.
 4. The method of claim 3, wherein administration of theextracellular matrix digesting enzyme and botulinum toxin to the bladderluminal surface area is achieved by instillation of the extracellularmatrix digesting enzyme and botulinum toxin into a bladder and thesurface area is a bladder luminal surface area and the target includes adetrusor muscle.
 5. The method of claim 3, wherein administration of theextracellular matrix digesting enzyme is accomplished by injection ofthe first composition into a bladder wall of a bladder, andadministration of the second composition containing the botulinum toxinis by instillation into the bladder and the target includes a detrusormuscle.
 6. The method of claim 3, wherein administration of theextracellular matrix digesting enzyme is accomplished by instillation ofthe first composition into a bladder, and administration of thebotulinum toxin is accomplished by injection of the second compositioninto a bladder wall of the bladder and the target includes a detrusormuscle.
 7. The method of claim 6, wherein administration of the secondcomposition containing the botulinum toxin accomplished by less than 20injections into the bladder wall.
 8. The method of claim 6, whereinadministration of the second composition containing the botulinum toxinaccomplished by less than 10 injections into the bladder wall.
 9. Themethod of claim 6, wherein administration of the second compositioncontaining the botulinum toxin accomplished by performing between 1 and5 injections into the bladder wall.
 10. The method of claim 4, whereinthe botulinum toxin is selected from the group consisting of botulinumtoxin type A, B, C₁, D, E, F and G.
 11. The method of claim 4, furthercomprising the steps of emptying the bladder prior to step (a), emptyingthe bladder after step (b) and emptying the bladder after step (e). 12.The method of claim 2, wherein the cholinergically-influenced disorderis a urologic disorder selected from the group consisting of a bladderdisorder and a prostate disorder.
 13. The method of claim 12, whereinthe bladder disorder is selected from the group consisting of overactivebladder, hypertrophied bladder neck and detrusor hyperreflexia.
 14. Themethod of claim 12, wherein the prostate disorder is selected from thegroup consisting of benign prostatic hyperplasia, prostatitis andprostate cancer.
 15. The method of claim 1, further comprising the stepsof optionally removing the first composition after step (b) andoptionally removing the second composition after step (e).
 16. Themethod of claim 15, wherein the cholinergically-influenced disorder ishyperhidrosis and the surface area of the patient is selected from thegroup consisting of an axillary skin surface area, a palmar skin surfacearea and a plantar skin surface area.
 17. The method of claim 1, whereinthe extracellular matrix digesting enzyme is a member of the family ofhyaluronidases, tissue plasminogen activators and collagenases.
 18. Amethod for administering a neurotoxin to a patient in need thereof,wherein the method excludes injection of the neurotoxin or anextracellular matrix digesting enzyme, the method comprising the stepsof: a) administering a first composition containing at least oneextracellular matrix digesting enzyme onto a skin surface or luminalsurface of the patient; and b) administering the second compositioncontaining a neurotoxin onto the skin surface or luminal surface of thepatient, wherein the neurotoxin diffuses to a greater extent than ifadministered without the first composition containing at least oneextracellular matrix digesting enzyme, and wherein the administrationexcludes injection of both the first and second compositions.
 19. Themethod of claim 18, wherein the skin surface is an axillary skinsurface.
 20. The method of claim 18, wherein the neurotoxin is abotulinum toxin and is selected from the group consisting of botulinumtoxin types A, B, C₁, D, E, F and G.
 21. The method of claim 20, whereinthe administration of one or both of the extracellular matrix digestingenzyme and botulinum toxin is achieved by application via at least oneof spraying or rubbing onto the skin surface or luminal surface of thepatient.
 22. The method of claim 21, further comprising the step ofdrying the skin surface after step (a).
 23. The method of claim 22,wherein drying the skin surface comprises the step of allowingsufficient time to pass to allow evaporation of the first compositionfrom the skin surface, before commencing step (b).
 24. A non-injectionmethod for treating a urologic disorder in a patient in need thereof,comprising the steps of: a) instilling a first composition containinghyaluronidase into to a bladder of the patient in order to contact abladder luminal surface area, having a glycosaminoglycan layer, to thefirst composition; b) maintaining the first composition within thebladder to allow sufficient time to pass such that hyaluronidaseinteracts with the glycosaminoglycan layer and diffuses through thebladder luminal surface area; c) optionally draining the firstcomposition from the bladder; d) instilling a second compositioncontaining a botulinum toxin type A to the bladder in order to contactthe bladder luminal surface area previously contacted by the previouslyinstilled and optionally removed first composition; and e) retaining theinstilled second composition for a sufficient time within the bladder sothat a sufficient amount of botulinum toxin type A therein diffusesthrough the bladder luminal surface area and to at least one layer ofthe muscularis propria, and optionally, draining the second compositionfrom the bladder, thereby alleviating at least one symptom associatedwith the urologic disorder and treating the urologic disorder of thepatient in need thereof.
 25. The method of claim 24, wherein theurologic disorder is selected from the group consisting of overactivebladder, hypertrophied bladder neck and detrusor hyperreflexia.
 26. Amethod for administering a neurotoxin to a patient in need thereof,wherein the method excludes injection of the neurotoxin or anextracellular matrix digesting enzyme, the method comprising the stepof: administering a composition containing at least one extracellularmatrix digesting enzyme and a neurotoxin onto a skin surface or luminalsurface of the patient, wherein the neurotoxin diffuses to a greaterextent to or throughout a target than if administered without the firstcomposition containing at least one extracellular matrix digestingenzyme, and wherein the administration excludes injection of both thefirst and second compositions.
 27. The method according to claim 26,wherein the neurotoxin is a botulinum toxin and is selected from thegroup consisting of botulinum toxin types A, B, C₁, D, E, F and G. 28.The method according to claim 26, wherein the neurotoxin is a botulinumtoxin type A.
 29. The method according to claim 28, wherein the amountof botulinum toxin type A is between about 20 units and about 2750units.
 30. The method according to claim 27, wherein the at least oneextracellular matrix digesting enzyme is selected from the groupconsisting of a hyaluronidase, a tissue plasminogen activator and acollagenase.